| PurposeThe purpose of this study is to observe the heterogeneity of retinal tissue cells in patients with diabetes and analyze the expression of differential genes and the enrichment of functional signal pathways by using single-cell RNA sequencing(scRNA-seq)and verifying results in animal models.Laying a foundation for further analysis of the behavior of single-cell and their role and mechanism in the occurrence and development of diabetic retinopathy.MethodThe retinal tissues of donated eyeballs of 4 patients with type 2 diabetes mellitus(diabetic group)and 2 patients without diabetes mellitus(control group)were sequenced by a high throughput single-cell transcription group.The cell composition,differential genes,and expression of enrichment pathways between the two groups were compared by the results of standard analysis.Screening of cells,genes,and signal pathways that may play an important role in diabetic retinopathy by bioinformatics.Then,the above screening targets were preliminarily verified in the diabetic rat model.The diabetic rat model was established by a high-fat diet and intraperitoneal injection of streptozotocin(STZ).Untreated rats served as normal control.The eyeballs of rats were taken for frozen sections at the 2nd,4th,8th,and 12th week respectively,and the differences of selected cells and related molecules between the two experimental groups were compared after immunofluorescence staining.ResultThe two groups of human retinal samples were clustered and annotated to get 9 cell types:rods,cones,bipolar cells,Muller cells,microglia,amacrine cells,melanocytes,horizontal cells,and vascular cells.Bioinformatics analysis showed that the abundance of Müller cells in the diabetic group was higher than that in the control group,and the consistency was good within the group.Further analysis of the differential gene expression of Muller cells in the two groups showed that the expression of heat shock protein 90(HSP90)was significantly down-regulated in the diabetic group.The protein-protein interaction network based on differential genes in Muller cells was constructed by using STRING.It was found that HSP90 played the role of a core gene,in which the significant functional pathway was necroptosis.The results of animal experiments showed that the expression of the glial fibrillary acidic protein(GFAP),a marker of Müller cells in the retina of the diabetic group,increased with time,and was higher than that of the control group at each time point(t=-2.01,P<0.05;t=-5.72,P<0.001;t=-15.97,P<0.001;t=-16.78,P<0.001).The expression of HSP90 in the retina of the diabetic group was higher than that of the control group at 2 weeks(t=-5.46,P<0.001)and lower than that of the control group at 4 weeks,8 weeks,and 12 weeks(t=5.13,P<0.001;t=11.27,P<0.001;t=13.58,P<0.001).The co-expression of GFAP and HSP90 showed that the expression of HSP90 in the retinal Müller cells of the diabetic group was higher than that of the control group at 2 weeks(t=-11.75,P<0.001),and lower than that of the control group at 4 weeks,8 weeks and 12 weeks(t=4.45,P<0.001;t=10.49,P<0.001;t=12.39,P<0.001).ConclusionIn this study,the map of the single-cell transcriptome of retinal tissue in patients with diabetes mellitus was drawn for the first time.Bioinformatics analysis showed that significant heterogeneity of Müller cells suggested that they played a more diverse role in the pathological process of diabetic retinopathy.The significantly down-regulated HSP90 gene plays a core role in the protein interaction network based on differential genes in Müller cells,suggesting that HSP90 is expected to become an important regulatory gene with high activity in diabetic retinopathy.Whether programmed necrosis of the enrichment pathway is the functional pathway of the HSP90 gene remains to be verified by further experiments. |
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